CN110229857A - The preparation method of bird's nest peptide and its depolymerization coupled enzymatic - Google Patents
The preparation method of bird's nest peptide and its depolymerization coupled enzymatic Download PDFInfo
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- CN110229857A CN110229857A CN201910406255.0A CN201910406255A CN110229857A CN 110229857 A CN110229857 A CN 110229857A CN 201910406255 A CN201910406255 A CN 201910406255A CN 110229857 A CN110229857 A CN 110229857A
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- nest
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- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 7
- 238000002360 preparation method Methods 0.000 title claims description 43
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- 238000012449 Kunming mouse Methods 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides the methods that depolymerization coupled enzymatic prepares bird's nest peptide to crush the bird's nest after denaturation including being denaturalized to the bird's nest impregnated, smashed bird's nest is adjusted into pH to 6.5-7.5, protease is then added, is digested, to enzymolysis product enzyme deactivation, filtering.The bird's nest peptide prepared the present invention also provides this method and its application in drug or health food etc..
Description
Technical field
The invention belongs to protein and peptide technical fields, specifically, the present invention relates to be related to the depolymerization coupling of bird's nest albumen
The bird's nest peptide for closing enzymolysis preparation and its preparing.
Background technique
Bird's nest (Edible bird's nest, EBN) refers to that a variety of esculent swifts of Apodidae are solidifying with saliva and a small amount of down etc.
Tie built nest.Bird's nest is a kind of rare tonic, has nourishing yin to moisten dryness, tonifying middle-Jiao and Qi, enters the function such as stomach tonifying spleen, preventing phlegm from forming and stopping coughing
Can, it is known as " first of eight delicacies ".Chinese medicine thinks bird's nest: nourishing yin to moisten dryness, invigorating middle warmer and replenishing QI, governance wasting, expectoration asthma, hemoptysis, protracted dysentery are fitted
It is suitable for and has a delicate constitution, malnutritive, protracted dysentery chronic malaria, many coughs of phlegm, senile chronic bronchitis, bronchiectasis, pulmonary emphysema, lung
Tuberculosis, hemoptysis are spitted blood and stomachache patients.Modern medicine discovery, bird's nest can promote immune function, there is delaying human body caducity, prolong
The effect of year lengthens one's life.The study found that bird's nest main component has: water soluble protein, carbohydrate, microelement (including calcium,
Phosphorus, iron, sodium, potassium) and to the amino acid for promoting human activity to play an important role, wherein protein content is 60% or so.
Currently, the peptides product of some bird's nests is reported.For example, Chinese patent application CN107974479A is disclosed
A kind of method that bird's nest oligopeptide is prepared with microwave and membrane technology, comprising: first bird's nest is crushed, is impregnated with hot deionized water swollen
Hair, is starched with colloid mill circular grinding at bird's nest, adds deionized water, is adjusted pH value to 10.6, is flowed out protein with microwave treatment,
Assemble bird's nest oligopeptide in microwave-assisted lower carry out double-enzyme hydrolysis, then through ultrafiltration and nanofiltration.The peptide fragment of the oligopeptide is distributed in 5
- 16 peptide of peptide, the relative molecular weight of peptide are 539-1745Da.
For another example, Chinese patent application CN108070018A discloses a kind of method that bird's nest native peptides are extracted from bird's nest,
This method obtains the optimum condition for extracting bird's nest native peptides by screening test, is the pH value of 9.5, termination including initial pH value
It is≤8.4 etc..The peptide fragment for the bird's nest native peptides that this method is extracted is from 5 peptides to 19 peptides.
Also such as, Chinese patent application CN108893511A discloses a kind of method that bird's nest extracts small-molecular peptides, comprising: uses
Clear water impregnates bird's nest and drying;Bird's nest after drying is crushed, screening;Powder after screening is subjected to broken wall, obtains fine powder;To
Fine powder adds deionized water, is digested using zytase, pectase, papain complex gradient, then through papain
Protein is digested.
In addition, Chinese patent application CN107488225A discloses a kind of method that bird's nest extracts small-molecular peptides, comprising: will
Bird's nest is dried by the sun, is ground into powder, and in the powder plus deionized water is modulated into mixed liquor, and mixed liquor is put into ultrasonic water
Electromagnetic heating enzymatic hydrolysis is carried out after vibrating in bath under alkaline environment, with the centrifuge for the centrifugal drum for being mounted with 3 layers of filter membrane of band
Separation mixed liquor obtains refined filtration liquid, and the refined filtration liquid is placed in the cold low-temperature stabilization of icebox, refined filtration liquid is moved to vacuum drier
The small-molecular peptides that the bird's nest extracts are obtained after drying, improve human body to the absorbability of bird's nest small-molecular peptides.
The method of the peptides product of these prior art preparation bird's nests, what is had is cumbersome, and equipment investment is big, and what is had is simple and crude, produces
Rate is low, in addition to this, more seriously, although mostly brag about will be in bird's nest for the peptides product of the bird's nest of these technologies preparation
Breaks down proteins be the small-molecular peptides easily absorbed, improve the bioavailability of bird's nest, but they are actual medicinal or protect
Strong effect is inaccurate, it is difficult to practical to promote the use of.
The present inventor relies on long-term further investigation, has investigated a kind of depolymerization coupled enzymatic system to bird's nest unexpectedly
The method of standby bird's nest peptide, not only method is easy to operate, and equipment investment is little, and the new bird's nest peptide prepared with this method in addition to
Have the characteristics that small-molecular peptides easily absorb, and it is medicinal, health-care effect is definite, immunity can be significantly improved.
Summary of the invention
It is medicinal, health-care effect is definite the technical problem to be solved in the present invention is that provide new bird's nest peptide, better than tradition
The bird's nest product of technology preparation.In addition, improving the present invention also provides the preparation method of the bird's nest peptide and such as immunity side
The application in face etc..
Specifically, in a first aspect, coupling enzyme by the depolymerization included the following steps the present invention provides bird's nest peptide
Solve preparation method preparation:
(1) clean bird's nest is taken, is soaked in water;
(2) bird's nest impregnated is denaturalized;
(3) bird's nest after denaturation is crushed;
(4) smashed bird's nest is adjusted into pH to 6.5-7.5, protease is then added, is digested;
(5) to enzymolysis product enzyme deactivation, filtering;With
(6) optionally the enzymolysis liquid of filtering is concentrated, dried.
It is preferred that the weight ratio of bird's nest and water is 1:40- in the preparation step (1) of the bird's nest peptide of first aspect present invention
80, preferably 1:50-70, such as 1:60.
It is also preferred that the temperature of immersion is 15-30 DEG C, excellent in the preparation step (1) of the bird's nest peptide of first aspect present invention
It is selected as 22-26 DEG C, such as room temperature.In general, room temperature can be 25 DEG C.
Further preferably in the preparation step (1) of the bird's nest peptide of first aspect present invention, the time of immersion is 2-6 hours, excellent
It is selected as 3-5 hours, such as 4 hours.
The method being denaturalized in the preparation step (2) of the bird's nest peptide of first aspect present invention can be physical method, such as heating,
Pressurization, dehydration, stirring, oscillation, ultraviolet light irradiation and/or ultrasonication etc., are also possible to chemical method, such as with strong acid, strong
Alkali, urea, heavy metallic salt and/or lauryl sodium sulfate (SDS) etc. processing etc., can also be it is above-mentioned either physically or chemically in
Two or more combination.In a specific embodiment of the invention, denaturation is heat denatured.
It is preferred that the temperature of heating is 90-100 DEG C, preferably in the preparation step (2) of the bird's nest peptide of first aspect present invention
It is 95-100 DEG C, such as 100 DEG C.
It is also preferred that the time of heating is 30-60 minutes in the preparation step (2) of the bird's nest peptide of first aspect present invention,
Preferably 35-50 minutes, such as 40 minutes.
The method crushed in the preparation step (3) of the bird's nest peptide of first aspect present invention can be ultramicro grinding, colloid mill
It crushes, broken wall crushes, ball mill crushes, one or several kinds of combinations of homogenizer homogeneous and/or high pressure homogenizer.In this hair
In bright specific embodiment, crushing is homogenate.
It is preferred that adjusting the reagent of pH in the preparation step (4) of the bird's nest peptide of first aspect present invention as acid, preferably vinegar
Acid.
It is also preferred that protease is pepsin, tryptose in the preparation step (4) of the bird's nest peptide of first aspect present invention
Enzyme, animal proteolytic enzyme, neutral proteinase, papain, bromelain, alkali protease, stromatin enzyme, flavor
One of enzyme or two or more combinations.In a specific embodiment of the invention, protease is animal proteolytic enzyme
And/or papain.
Further preferably in the preparation step (4) of the bird's nest peptide of first aspect present invention, the temperature of enzymatic hydrolysis is 25-60 DEG C, excellent
It is selected as 30-55 DEG C, such as 50 DEG C.
In addition preferably in the preparation step (4) of the bird's nest peptide of first aspect present invention, the time of enzymatic hydrolysis is 1-6 hours,
Preferably 2-5 hours, such as 3 hours.
It is preferred that the temperature of enzyme deactivation is 80-100 DEG C, preferably in the preparation step (5) of the bird's nest peptide of first aspect present invention
It is 85-98 DEG C, such as 95 DEG C.
It is also preferred that the time of enzyme deactivation is 15-60 minutes in the preparation step (5) of the bird's nest peptide of first aspect present invention,
Preferably 20-45 minutes, such as 30 minutes.
Further preferably in the preparation step (5) of the bird's nest peptide of first aspect present invention, the membrane aperture of filtering is 150-200
Mesh, preferably 180-200 mesh, such as 200 mesh.
Herein, it optionally indicates it can be selected that can not also select.That is, the system of the bird's nest peptide of first aspect present invention
Standby step (6) can be not selected progress, then the bird's nest peptide of first aspect present invention exists in solution;Also it can choose
The step (6) is carried out, so that the bird's nest peptide of first aspect present invention exists in solid form.
The molecular particle size of the bird's nest peptide of first aspect present invention is small, is uniformly dispersed, and predominantly contains 2-20 amino acid
The polypeptide of residue.It is preferred that the content of small-molecular peptides of the molecular weight of the bird's nest peptide of first aspect present invention between 200-2500Da
For 70-90% (w/w), preferably 80-90% (w/w), for example 86-88% (w/w).
In second aspect, the present invention provides pharmaceutical compositions comprising the bird's nest peptide and pharmacy of first aspect present invention
Upper acceptable auxiliary material.In a specific embodiment of the invention, the pharmaceutical composition of second aspect of the present invention can be used for restoring
Impaired immune organ improves abdominal cavity macrophage phagocytosis.
Herein, term " pharmaceutically acceptable auxiliary material " includes pharmaceutically acceptable carrier, excipient, diluent
Deng they are compatible with active pharmaceutical ingredient.Pharmaceutical preparation is prepared to ordinary skill with pharmaceutically acceptable auxiliary material
It is well known for personnel.Pharmaceutical composition of the invention includes the bird's nest peptide of first aspect present invention as active constituent, will
The bird's nest peptide and pharmaceutically acceptable adjuvant (carrier, excipient, diluent as would be known to one of ordinary skill in the art
Deng) combine, various preparations, preferably solid pharmaceutical preparation and liquid preparation are configured to, as tablet, pill, capsule (including are held
Releasing pattern is released in continuous release or delay), pulvis, suspension, granule, tincture, syrup, emulsion agent, the dosage forms such as suspension with
And various sustained-release dosage types, to be suitble to various form of medication, such as form of medication oral or by skin etc., most preferably mouth
Clothes.
In the third aspect, the present invention provides health-care food compositions comprising the bird's nest peptide of first aspect present invention and
Acceptable auxiliary material in health food.In a specific embodiment of the invention, the health food combination of third aspect present invention
Object can be used for restoring impaired immune organ, improve abdominal cavity macrophage phagocytosis.
Herein, term " acceptable auxiliary material in health food " include acceptable carrier on food, it is excipient, dilute
Agent, flavoring agent, colorant, fumet etc. are released, they are compatible with the healthcare active of health food.First aspect present invention
Bird's nest peptide can be directly added into food perhaps can for example add in raw-food material contain or soup without containing sweetener or
In custard.
In fourth aspect, the present invention provides the methods that depolymerization coupled enzymatic prepares bird's nest peptide comprising following steps:
(1) clean bird's nest is taken, is soaked in water;
(2) bird's nest impregnated is denaturalized;
(3) bird's nest after denaturation is crushed;
(4) smashed bird's nest is adjusted into pH to 6.5-7.5, protease is then added, is digested;
(5) to enzymolysis product enzyme deactivation, filtering;With
(6) optionally the enzymolysis liquid of filtering is concentrated, dried.
It is preferred that the weight ratio of bird's nest and water is 1:40-80, preferably the method for fourth aspect present invention the step of in (1)
For 1:50-70, such as 1:60.
It is also preferred that the temperature of immersion is 15-30 DEG C, preferably 22- the method for fourth aspect present invention the step of in (1)
26 DEG C, such as room temperature.In general, room temperature can be 25 DEG C.
Further preferably the method for fourth aspect present invention the step of in (1), the time of immersion is 2-6 hours, preferably 3-5
Hour, such as 4 hours.
The method being denaturalized in the step of method of fourth aspect present invention (2) can be physical method, such as heat, pressurize,
Dehydration, stirring, oscillation, ultraviolet light irradiation and/or ultrasonication etc., are also possible to chemical method, such as use strong acid, highly basic, urine
Processing such as element, heavy metallic salt and/or lauryl sodium sulfate (SDS) etc., can also be it is above-mentioned either physically or chemically in two kinds
Or two or more combination.In a specific embodiment of the invention, denaturation is heat denatured.
It is preferred that the temperature of heating is 90-100 DEG C, preferably 95- the method for fourth aspect present invention the step of in (2)
100 DEG C, such as 100 DEG C.
It is also preferred that the time of heating is 30-60 minutes, preferably the method for fourth aspect present invention the step of in (2)
35-50 minutes, such as 40 minutes.
The method crushed in the step of method of fourth aspect present invention (3) can be ultramicro grinding, colloid mill crushes, broken
Wall crushes, ball mill crushes, one or several kinds of combinations of homogenizer homogeneous and/or high pressure homogenizer.In tool of the invention
In body embodiment, crushing is homogenate.
It is preferred that adjusting the reagent of pH the method for fourth aspect present invention the step of in (4) as acid, preferably acetic acid.
It is also preferred that the method for fourth aspect present invention the step of in (4), protease is pepsin, trypsase, dynamic
Object proteolytic enzyme, neutral proteinase, papain, bromelain, alkali protease, stromatin enzyme, in food flavor enzyme
One or more kinds of combinations.In a specific embodiment of the invention, protease be animal proteolytic enzyme and/or
Papain.
Further preferably the method for fourth aspect present invention the step of in (4), the temperature of enzymatic hydrolysis is 25-60 DEG C, preferably 30-
55 DEG C, such as 50 DEG C.
In addition preferably the method for fourth aspect present invention the step of in (4), the time of enzymatic hydrolysis is 1-6 hours, preferably
2-5 hours, such as 3 hours.
It is preferred that the temperature of enzyme deactivation is 80-100 DEG C, preferably 85- the method for fourth aspect present invention the step of in (5)
98 DEG C, such as 95 DEG C.
It is also preferred that the time of enzyme deactivation is 15-60 minutes, preferably the method for fourth aspect present invention the step of in (5)
20-45 minutes, such as 30 minutes.
Further preferably the method for fourth aspect present invention the step of in (5), the membrane aperture of filtering is 150-200 mesh, preferably
For 180-200 mesh, such as 200 mesh.
At the 5th aspect, the present invention provides the bird's nest peptides of first aspect present invention to improve or restore immunity in preparation
Application in drug or health food.In a specific embodiment of the invention, the bird's nest peptide of first aspect present invention can be used for
Restore impaired immune organ, improve abdominal cavity macrophage phagocytosis, restores or the ability of raising is significantly better than and is made in the conventional way
Bird's nest product.
It is preferred that drug is the pharmaceutical composition of second aspect of the present invention in the application of fifth aspect present invention.
It is also preferred that health food is the health food combination of third aspect present invention in the application of fifth aspect present invention
Object.
The beneficial effects of the present invention are: provide new bird's nest peptide, it is medicinal, health-care effect is definite, it can significantly improve
Immunity, significant effect are better than the bird's nest product of conventional method preparation;The bird's nest peptide prepares bird's nest peptide using depolymerization coupled enzymatic
Method preparation, preparation method is easy to operate, and equipment investment is little.
In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.It needs to refer in particular to
Out, these descriptions are only exemplary description, and are not meant to limit the scope of the invention.Opinion according to this specification
It states, many variations of the invention, change will be apparent from for those skilled in the art.
In addition, the present invention refers to open source literature, these documents are their full text in order to more clearly describe the present invention
Content is included in and is referred to herein, just looks like that repeated description herein has been excessively for their full text.
Specific embodiment
The contents of the present invention are further illustrated by the following examples.Such as not specified, technology used in embodiment
The conventional means and commercially available common instrument, reagent that means are well known to those skilled in the art, reference can be made to " Molecular Cloning: A Laboratory
Guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and
The reference such as manufacturers instruction of reagent.
The illustrative preparation method 1 of 1 bird's nest peptide of embodiment
Dry bird's nest (the white swallow of Malaysia) 6.5g is weighed, 60 times of (relative to dry bird's nest) purifying are added after cleaning up
Water soaking at room temperature 4 hours.The bird's nest impregnated is placed in closed container together with water, is divided with heat denatured 40 in 100 DEG C of boiling water baths
Clock is subsequently cooled to 50 DEG C.By after denaturation bird's nest be packed into refiner (MA60 model is purchased from OuHor company) with 3000 turns/
Second homogeneous is homogenized 5 minutes.Dilute hydrochloric acid is added to the bird's nest of homogenate, adjusts pH to 7,13mg papain (200,000 is then added
U/g enzyme activity, is purchased from Pangbo Bioengineering Co Ltd, Nanning) and 13mg alkali protease (200,000 u/g enzyme activities, it is commercially available
From Pangbo Bioengineering Co Ltd, Nanning), in 50 DEG C constant-temperature table digestion 3 hours.Enzymolysis product is heated to 95 DEG C, is kept
30 minutes, enzyme is inactivated, then, crosses the removal of impurities of 200 mesh filter clothes, obtain clarification enzymolysis liquid.Finally, enzymolysis liquid is concentrated, it is freeze-dried
Obtain 6.1 grams of bird's nest Gly-His-Lys.
It is detected through GPC gel permeation chromatography, small molecule of the resulting bird's nest Gly-His-Lys middle-molecular-weihydroxyethyl between 200-2500Da
The content of peptide is 87%.
The illustrative preparation method 2 of 2 bird's nest peptide of embodiment
The method that preparation method is substantially identical to embodiment 1, except that with 13mg animal proteolytic enzyme (200,000 u/
G enzyme activity, is purchased from Pangbo Bioengineering Co Ltd, Nanning) replace papain to be digested.Finally obtain bird's nest peptide
6.0 grams of powder, through detecting, the content of small-molecular peptides of the resulting bird's nest Gly-His-Lys middle-molecular-weihydroxyethyl between 200-2500Da is 80%.
Bird's nest peptide preparation method of the comparative example 1 without crushing process
The method that preparation method is substantially identical to embodiment 1, except that the bird's nest after denaturation is not packed into refiner, and
It is directly to adjust pH after being cooled to 50 DEG C, adds papain enzymolysis and alkali protease, and carry out subsequent step.It finally obtains
6.0 grams of bird's nest Gly-His-Lys, through detecting, the content of small-molecular peptides of the resulting bird's nest Gly-His-Lys middle-molecular-weihydroxyethyl between 200-2500Da is
63%.
Bird's nest peptide preparation method of the comparative example 2 without degenerative process
The method that preparation method is substantially identical to embodiment 1, except that the bird's nest impregnated without denaturation, but
It is directly loadable into refiner homogenate, and carries out subsequent step.6.0 grams of bird's nest Gly-His-Lys are finally obtained, through detecting, resulting bird's nest Gly-His-Lys
The content of small-molecular peptides of the middle-molecular-weihydroxyethyl between 200-2500Da is 55%.
The traditional preparation methods of 3 bird's nest peptide of comparative example
Dry bird's nest 6.5g is weighed, it is small that 60 times of (relative to dry bird's nest) purified water soaking at room temperature 4 are added after cleaning up
When.The bird's nest impregnated is placed in closed container, is stewed 12 hours with 95 DEG C of high temperature, is then crossed 200 mesh filter clothes, obtain clarification filtration
Liquid.Finally, filtered fluid is concentrated, freeze-drying obtains 5.9 grams of bird's nest Gly-His-Lys.
It is detected through GPC gel permeation chromatography, small molecule of the resulting bird's nest Gly-His-Lys middle-molecular-weihydroxyethyl between 200-2500Da
The content of peptide is 43%.
Effect of the effect example bird's nest peptide to immune function of mice
Using the bird's nest peptide of according to embodiments of the present invention and comparative example preparation, to observe the effect to immune function of mice,
Specific experiment is as follows:
1, experimental method
70 kunming mices are taken, are randomly divided into following 7 groups, every group 10, the drug of the daily following each group of gastric infusion, often
It is primary, and each 0.1ml is carried out continuously 4 weeks:
(1) normal group: physiological saline
(2) model group: physiological saline
(3) bird's nest peptide group one: the bird's nest peptide of 1 preparation according to embodiments of the present invention, dosage 20mg/kg
(4) bird's nest peptide group two: the bird's nest peptide of 2 preparation according to embodiments of the present invention, dosage 20mg/kg
(5) bird's nest peptide group three: the bird's nest peptide that according to the present invention prepared by comparative example 1, dosage 20mg/kg
(6) bird's nest peptide group four: the bird's nest peptide that according to the present invention prepared by comparative example 2, dosage 20mg/kg
(7) bird's nest peptide group five: the bird's nest peptide that according to the present invention prepared by comparative example 3, dosage 20mg/kg.
In addition to Normal group, remaining each group mouse subcutaneous injection hydrocortisone on the 21st after first administration,
Dosage is 15mg/kg, once a day, is carried out continuously 6 days.After the last administration, mouse is deprived of food but not water 12 hours, every mouse abdomen
Chamber is weighed after injecting 2% chicken erythrocyte suspension 1mL, 30min, and then rear cervical dislocation is put to death, and removes spleen, thymus gland weighing respectively,
To calculate spleen index and thymus index;In addition peritoneal macrophage is collected, is dyed and is counted, phagocytic rate is calculated.
2, experimental result
The results are shown in Table 1 for the spleen index and thymus index of each group mouse, wherein the spleen index and thymus gland of model group refer to
Number is substantially less than normal group, shows that the ratio of immune organ and weight can be substantially reduced by continuously injecting hydrocortisone, be shown
Modeling success;Bird's nest peptide group five conventionally can slightly improve spleen index and thymus index, however relative to model group,
It is this to improve no conspicuousness;According to the bird's nest peptide group one and group two of the method for the present invention spleen can be significantly improved relative to model group
Index and thymus index;The bird's nest peptide group three and group four for cannoing be used up whole method improve the amplitude of spleen indexs and thymus index between
Conventionally and according between the method for the present invention, the thymus index of raising is significantly higher than model group, but spleen index mentions
Height does not have conspicuousness relative to model group.
Influence of each drug of table 1 to the spleen index and thymus index of mouse
Group | Spleen index | Thymus index |
Normal group | 4.43±0.41 | 1.53±0.37 |
Model group | 3.11±0.30 | 1.15±0.28 |
Bird's nest peptide group one | 3.80±0.33 | 1.91±0.31 |
Bird's nest peptide group two | 3.72±0.36 | 1.85±0.22 |
Bird's nest peptide group three | 3.57±0.52 | 1.69±0.29 |
Bird's nest peptide group four | 3.64±0.55 | 1.72±0.25 |
Bird's nest peptide group five | 3.42±0.46 | 1.59±0.39 |
The result shows that can significantly restore impaired immune organ according to bird's nest peptide prepared by the method for the present invention;Even if saving
Slightly a part of step of the method for the present invention, the bird's nest peptide of preparation also can significantly restore impaired partial immunity organ, restore width
Degree is prepared not as good as the method for the present invention;Although and the bird's nest peptide traditionally prepared can restore from mean value, group
On do not show and restore in the presence of significant.
The results are shown in Table 2 for the abdominal cavity macrophage phagocytosis of each group mouse, wherein the abdominal cavity macrophage phagocytosis of model group is aobvious
It writes and is lower than normal group, show that macrophages phagocytic capacity can be significantly reduced by continuously injecting hydrocortisone, show modeling success;Nothing
By whole method conventionally, is cannotd be used up, also according to the method for the present invention, prepared bird's nest peptide can be relative to model
Group significantly improves abdominal cavity macrophage phagocytosis, wherein mentioning according to the increase rate maximum of the method for the present invention, and conventionally
High-amplitude is minimum.
Influence of each drug of table 2 to the macrophages phagocytic capacity of mouse
Group | Abdominal cavity macrophage phagocytosis (%) |
Normal group | 33.6±5.1 |
Model group | 24.3±4.7 |
Bird's nest peptide group one | 42.5±3.9 |
Bird's nest peptide group two | 40.8±2.8 |
Bird's nest peptide group three | 36.4±3.5 |
Bird's nest peptide group four | 37.5±5.3 |
Bird's nest peptide group five | 33.4±4.4 |
The result shows that the bird's nest peptide prepared using various methods can significantly improve the macrophages phagocytic capacity of mouse,
Promote immune function, and it is best according to effect prepared by the method for the present invention.
Claims (10)
1. bird's nest peptide is prepared by the depolymerization coupled enzymatic preparation method included the following steps:
(1) clean bird's nest is taken, is soaked in water;
(2) bird's nest impregnated is denaturalized, preferably heat denatured;
(3) bird's nest after denaturation is crushed, is preferably homogenized;
(4) smashed bird's nest is adjusted into pH to 6.5-7.5, protease is then added, is digested;
(5) to enzymolysis product enzyme deactivation, filtering;With
(6) optionally the enzymolysis liquid of filtering is concentrated, dried.
2. bird's nest peptide described in claim 1, wherein
The weight ratio of bird's nest and water in step (1) is 1:40-80, preferably 1:50-70, such as 1:60;
The temperature of immersion in step (1) be 15-30 DEG C, preferably 22-26 DEG C, such as room temperature;And/or
The time of immersion in step (1) is 2-6 hours, preferably 3-5 hours, such as 4 hours.
3. bird's nest peptide described in claim 1, wherein
The temperature of heating in step (2) is 90-100 DEG C, preferably 95-100 DEG C, such as 100 DEG C;And/or
The time of heating in step (2) is 30-60 minutes, preferably 35-50 minutes, such as 40 minutes.
4. bird's nest peptide described in claim 1, wherein
The reagent of adjusting pH in step (4) is acid, preferably acetic acid;
Protease in step (4) is pepsin, trypsase, animal proteolytic enzyme, neutral proteinase, Papain
One of enzyme, bromelain, alkali protease, stromatin enzyme, food flavor enzyme or two or more combinations, it is preferably dynamic
Object proteolytic enzyme and/or papain;
The temperature of enzymatic hydrolysis in step (4) is 25-60 DEG C, preferably 30-55 DEG C, such as 50 DEG C;And/or
The time of enzymatic hydrolysis in step (4) is 1-6 hours, preferably 2-5 hours, such as 3 hours.
5. bird's nest peptide described in claim 1, wherein
The temperature of enzyme deactivation in step (5) is 80-100 DEG C, preferably 85-98 DEG C, such as 95 DEG C;
The time of enzyme deactivation in step (5) is 15-60 minutes, preferably 20-45 minutes, such as 30 minutes;And/or
The membrane aperture of filtering in step (5) is 150-200 mesh, preferably 180-200 mesh, such as 200 mesh.
6. bird's nest peptide described in claim 1, the content of small-molecular peptides of the middle-molecular-weihydroxyethyl between 200-2500Da is 70-
90% (w/w), preferably 80-90% (w/w), for example 86-88% (w/w).
7. pharmaceutical composition comprising any the bird's nest peptide and pharmaceutically acceptable auxiliary material of claim 1-6.
8. health-care food composition comprising acceptable in any the bird's nest peptide and health food of claim 1-6
Auxiliary material.
9. the method that depolymerization coupled enzymatic prepares bird's nest peptide comprising following steps:
(1) clean bird's nest is taken, is soaked in water;
(2) bird's nest impregnated is denaturalized, preferably heat denatured;
(3) bird's nest after denaturation is crushed, is preferably homogenized;
(4) smashed bird's nest is adjusted into pH to 6.5-7.5, protease is then added, is digested;
(5) to enzymolysis product enzyme deactivation, filtering;With
(6) optionally the enzymolysis liquid of filtering is concentrated, dried.
10. any bird's nest peptide of claim 1-6 improves in preparation or restores in the drug or health food of immunity
Application.
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